Palmarumycin based inhibitors of thioredoxin and methods of using same

ABSTRACT

Embodiments of the present invention relate to inhibitors of thioredoxin. Certain embodiments relate to palmarumycin based compounds and methods of using the same. Such compounds may be useful in inhibiting the overexpression of thioredoxin, inhibiting tumor growth and treating cancer.

This application claims priority to U.S. Provisional Application No.60/717,398 filed Sep. 15, 2005 titled “PALMARUMYCIN BASED COMPOUNDS ANDMETHODS OF USING SAME” with the United States Patent and TrademarkOffice, the contents of which are incorporated herein by reference.

GOVERNMENT INTERESTS

The United States Government may have certain rights to this inventionpursuant to work funded under NIH grants CA52995 and CA9082 1.

BACKGROUND

The thioredoxin redox couple thioredoxin/thioredoxin reductase (TR/Trx)is a ubiquitous redox system found in both prokaryotic and eukaryoticcells. The thioredoxin system is comprised primarily of two elements:thioredoxin and thioredoxin reductase. Thioredoxins are a class of lowmolecular weight redox proteins characterized by a highly conservedCys-Gly-Pro-Cys-Lys active site. The cysteine residues at the activesite of thioredoxin undergo reversible oxidation-reduction catalyzed bythioredoxin reductase. Trx-1 is ubiquitously expressed with a conservedcatalytic site that undergoes reversible NADPH-dependent reduction byselenocysteine-containing flavoprotein Trx-1 reductases.

The cytosolic thioredoxin redox system is composed of thioredoxin-1 andthioredoxin reductase-1 reductase, which catalyzes the NADPH-dependentreduction of thioredoxin-1. Thioredoxin reductase-1 is an importantregulator of cancer cell growth and survival. Thioredoxin-1 acting withperoxiredoxin-1 is an antioxidant that scavenges 11202. Thioredoxins arealso able to reduce buried oxidized thiol residues in proteins andregulate the activity of redox-sensitive transcription factors,including p53, nuclear factor-nB, the glucocorticoid receptor, activatorprotein-1, hypoxia-inducible factor-1 (HIF-1), Sp1, and Nrf2.Thioredoxin-1 also binds and inhibits the activity of the apoptosisinducing proteins, apoptosis signal-regulating kinase-1 and, the tumorsuppressor phosphatase and tensin homologue deleted on chromosome 10,thus inhibiting apoptosis. Thioredoxin-1 is overexpressed in many humantumors where it is associated with increased cell proliferation,decreased apoptosis, and poor patient survival. Thioredoxin reductasethus provides a target to regulate the activity of overexpressedthioredoxin-1.

Thioredoxin reductase-1 is a selenocysteine-containing flavoprotein withbroad substrate specificity because of the ready accessibility of itsCOOH-terminal redox active site, which contains an essentialselenocysteine residue. There are three thioredoxin reductase isoforms:the canonical cytoplasmic thioredoxin reductase-1, a mitochondrialthioredoxin reductase-2, and a testes-specific thioredoxinreductase/glutathione reductase. The cellular level of thioredoxinreductase-1 is subject to complex regulation. The core promoter of thethioredoxin reductase-1 gene contains several transcription factoractivation sites, including those for the redox-sensitive factors Oct-1and Sp1 as well as others. Differential splicing and alternativetranscription start sites result in multiple forms of the enzyme.Post-transcriptional regulation involving a selenocysteine insertionsequence element in the 3V-untranslated region directs selenocysteineincorporation, which is necessary for enzyme activity; thus, seleniumsupplementation can lead to increased thioredoxin reductase-1 activityin cell culture and in selenium deficient animals. Thioredoxinreductase-1 is necessary for cell proliferation. A thioredoxinreductase-1 knockout is embryonic lethal in mice, and thioredoxinreductase-1-deficient fibroblasts derived from the thioredoxinreductase-1 (−/−) embryos do not proliferate in vitro. Furthermore,cancer cell growth is inhibited by thioredoxin reductase-1 antisense,thioredoxin reductase-1 small interfering RNA and by a mutant redoxinactive thioredoxin reductase-1. There are reports that levels ofthioredoxin reductase-1 are increased by epidermal growth factor andhypoxia in cancer cells, although tumors show only moderately increasedlevels of thioredoxin reductase-1.

The redox protein thioredoxin-1 (Trx-1) has been proven to be a rationaltarget for anticancer therapy involved in promoting both proliferationand angiogenesis, inhibiting apoptosis, and conferring chemotherapeuticdrug resistance. Trx-1 was originally studied for its ability to act asa reducing cofactor for ribonucleotide reductase, the first unique stepin DNA synthesis. Thioredoxin also exerts specific redox control over anumber of transcription factors to modulate their DNA binding and, thus,to regulate gene transcription. Transcription factors regulated bythioredoxin include, but are not limited to, NF-κβ, p53, TFIIIC, BZLF1,the glucocorticoid receptor, and hypoxia inducible factor 1α (HIF-1α).Trx-1 also binds in a redox-dependent manner and regulates the activityof enzymes such as apoptosis signal-regulating kinase-1 protein kinasesC δ,{acute over (ε)}, ξ, and the tumor suppressor phosphatase PTEN.Trx-1 expression is increased in several human primary cancers,including, but not limited to, lung, colon, cervix, liver, pancreatic,colorectal, and squamous cell cancer. Clinically increased Trx-1 levelshave been linked to aggressive tumor growth, inhibition of apoptosis,and decreased patient survival.

Regulation of the thioredoxin-thioredoxin reductase (Trx-1/TrxR) systemis attracting increasing interest due to its implication in cancer,HIV-AIDS and rheumatoid arthritis along with other medical conditions.The naphthoquinone spiroketal pharmacophore of the palmarumycin familyof fungal metabolites holds promising biological activity against theTrx-1/TrxR system. Embodiments of the present invention relate tovarious analogues of the palmarumycin family and the ability of theseanalogues to inhibit the thioredoxin-thioreductase system.

SUMMARY OF THE INVENTION

Aspects of the present invention generally relate to analogs ofpalmarumycin. Such analogs may be effective in inhibitingthioredoxin/thioredoxin reductase (Trx/TrxR) system. Inhibition of theTrx/TrxR system may lead to inhibition of tumor growth. Therefore,further embodiments of the present invention provide compounds andpharmaceutical compositions for the inhibition of tumor growth.

Embodiments of the invention provide analogs of palmarumycin which canserve as lead compounds for the identification of a more efficaciousinhibitory compound. Further embodiments include use of the O-glycylnaphthoquinone spiroketal derivative of the palmarumycin as a leadcompound for the further development of Trx/TrxR system inhibitorycompounds. Embodiments of the invention also contemplate providing apalmarumycin derivative that may be cleaved to an active compound underphysiological conditions.

Embodiments of the invention also provide methods of inhibiting theTrx/TrxR system. Inhibition of the Trx/TrxR system may further inhibitvarious transcription factors, and subsequently promote apoptosis. Thus,further embodiments provide methods of inhibiting tumor growth in asubject in need of such treatment.

Embodiments of the invention relate to a compound, or salt thereof,having the general formula:

wherein R₁ may be H or OH;

R₂ may be OH, OCH₃, O(CH₂)nCH₃, OCH(CH₃)CH₂nCH₃, wherein n is 1-4,OCH₂CH₂-morpholino, OC(O)CH₂NH₂, OC(O)CH(CH₃) NH₂, OC(O)CH(CH(CH₃)₂)NH₂,OC(O)CH(CH₂Phenyl)NH₂, OC(O)CH(CH₂ p-OHPhenyl)NH₂

OC(O)CH(CH2OH)NH₂, OC(O)CH(CH₂SH)NH₂, OC(O)CH(CH₂COOH)NH₂,OC(O)CH(CH₂CH₂COOH)NH₂, OC(O)CH(CH₂CONH₂)NH₂, OC(O)CH(CH₂CH₂CONH₂)NH₂,OC(O)CH(CH(CH₃)CH₂CH₃)NH₂, OC(O)CH(CH₂CH(CH₃)₂)NH₂, orOC(O)CH(CH(OH)CH₃)NH₂;

and

R₃ may be a hydrogen, NHNHC(CH₃)₂CONH₂ or a carbon-carbon bond, with theproviso that if R₁ is OH and R₃ is a carbon-carbon bond, R₂ is not OH;or salts thereof. Exemplary salts include, but are not limited to HCl,TFA, tosylate or any other pharmaceutically acceptable salt.

In an alternative embodiment provided is a compound having the followingstructure:

In another alternative embodiment provided is a compound having thefollowing structure:

Preferred embodiments relate to a compound of the structure:

In a preferred embodiment, a salt of a palmarumycin compound has thestructure:

Other embodiments include compounds of the structures:

Preferred salts of the foregoing compounds include the following:

Embodiments further provide a compound derived from a palmarumycinwherein the compound inhibits a thioredoxin/thioredoxin reductasesystem. In one embodiment, the compound is a naphthoquinone spiroketalderivative. In another embodiment the compound is an O-glycylnaphthoquinone spiroketal derivative. Embodiments also provide anO-glycl naphthoquinone spiroketal derivative of palmarumycin.

Embodiments of the invention further provide a method of inhibiting athioredoxin/thioredoxin reductase system comprising contacting a cellwith a palmarumycin analog; and inhibiting the thioredoxin/thioredoxinreductase system. In one embodiment of the method, the palmarumycinanalog is an O-glycyl naphthoquinone spiroketal derivative.

An embodiment of the present invention provides a method of inhibitingtumor growth comprising administering a therapeutically effective amountof a palmarumycin analog to inhibit tumor growth. In preferredembodiments, the palmarumycin analog may be an O-glycl naphthoquinonespiroketal derivative of palmarumycin. The O-glycyl naphthoquinonespiroketal derivative may also be cleaved in vivo to an activepalmarumycin analog.

Another embodiment is a method for making a derivative of a palmarumycincomprising introducing a charged, hydrolytically cleavable function to anaphthoquinone spiroketal scaffold of palmarumycin to form a derivativeof palmarumycin.

In a further embodiment, a method for making a compound that inhibits athioredoxin/thioredoxin reductase system in vivo is provided. The methodcomprises identifying a compound as a lead compound, modifying the leadcompound, and selecting at least one analog of the lead compound thatexhibits inhibition of the thioredoxin/thioredoxin reductase system. Thelead compound may be palmarumycin. In another embodiment, the leadcompound may be an O-glycl naphthoquinone spiroketal derivative of thepalmarumycin.

DESCRIPTION OF THE DRAWINGS

In part, other aspects, features, benefits and advantages of theembodiments of the present invention will be apparent with regard to thefollowing description, appended claims and accompanying drawings where:

FIG. 1. Preparation of glycine and valine-derived prodrugs.

FIG. 2. Preparation of morpholine-derived prodrugs.

FIG. 3. Inhibition of cellular thioredoxin reductase by PX-916. MCF-7human breast cancer cells grown in medium containing 1 μM Se for 7 dayswere treated with PX-916 and total cellular thioredoxin reductaseactivity measured. A. Time course of the inhibition of the thioredoxinreductase on exposure to 1 μM PX-916. B. Concentration dependence of theinhibition of thioredoxin reductase on exposure to variousconcentrations of PX-916 for 17 hours. In both cases values are the meanof 3 separate determinations and bars are S.E. P≦0.04 or **p≦0.01. C.Inhibition of MCF-7 tumor thioredoxin reductase by PX-916. MCF-7 humanbreast cancer xenografts were grown in female scid mice implanted with17-β-estradiol 60 day slow-release pellets until they were ˜300 mm³. Themice were administered a single dose of PX-916 of 25 mg/kg i.v. andtumors harvested at various times. Thioredoxin reductase activity wasmeasured in tumor homogenates. Values are the mean of 3 mice at eachtime point and bars are SE. **p<0.01 compared to pretreatment value.

FIG. 4. Antitumor activity of PX-916. A. Female beige nude mice wereinoculated subcutaneously (s.c.) with 10⁷ A-673 rhabdomyosarcoma cells.When tumors were 100 mm³ on day 8 (arrow) dosing was begun with (∘)vehicle alone; (▴) PX-916 10 mg/kg intraperitoneally (i.p.) daily for 4doses. B. Female scid mice were inoculated s.c. with 10⁷ SHP-77 humansmall cell lung cancer cells. When tumors were 130 mm³ on day 17 (arrow)dosing was begun with (∘) vehicle alone; (▪) PX-916 10 mg/kg i.v. dailyfor 8 doses; (▴) PX-916 25 mg/kg i.v. daily for 5 doses. C. Female scidmice implanted 1 day previously with a 17-β-estradiol 60 day slowrelease pellet were inoculated s.c. with 10⁷ MCF-7 human breast cancercells. When tumors were 180 mm³ on day 8 (arrow) dosing was begun with(∘) vehicle alone; (⋄) PX-916 27.5 mg/kg i.v. daily for 5 doses. In bothA and B values are the mean of 8 mice per group and bars are SE.

FIG. 5. Inhibition of tumor HIF-1α, VEGF and thioredoxin reductase byrepeated administration of PX-916. Female scid mice were implanted 1dpreviously with a 90-d 17β-estradiol slow release pellet were inoculateds.c. with 10⁷ MCF-7 human breast cancer cells. When the tumors were 300mm³, vehicle or 25 mg/kg/d PX-916 was given i.v. for five doses. Twentyfour hours later, the tumors were removed and stained byimmunohistochemistry for HIF-1α and VEGF (A) or assayed for thioredoxinreductase activity (B). Columns mean of four mice, SE. P≦0.05.

FIG. 6. Stability of palmarumycin analogs in ethanol. (•) 1(eee269-111); (∘) 2A (eee86-111); (▴) 2B (eee86-111); (Δ) 3(eee-263-111); (▪) 4 (eee-273-11); and (□) SML-216.

FIG. 7. Stability of PX-916 and eee 263-111 in plasma.

FIG. 8. Stability of eee 86-11 and eee273-11 in plasma.

DESCRIPTION OF THE INVENTION

Embodiments of the invention relate to the generation of analogs ofpalmarumycin. Analogs are tested for their efficacy in inhibiting thethioredoxin/thioredoxin reductase system. Also, the analogs may be usedas lead compounds to identify further lead compounds and/or compoundsfor therapeutic use. The ability of these analogs to inhibit thethioredoxin—thioredoxin reductase system, both in vitro and in vivo, mayprovide beneficial and useful therapeutic agents. Embodiments of theinvention further relate to the anti-proliferative actions of thepalmarumycin analogues in tumor cells.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart. Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. As used herein, theterm “analog” or “derivative” are used interchangeably to mean achemical substance that is related structurally and functionally toanother substance. An analog or derivative contains a modified structurefrom the other substance, and maintains the function of the othersubstance, in this instance, inhibiting a thioredoxin/thioredoxinreductase. The analog or derivative need not, but can be synthesizedfrom the other substance. For example, a palmarumycin analog means acompound structurally related to palmarumycin, but not necessarily madefrom palmarumycin.

Optical Isomers—Diastereomers—Geometric Isomers-Tautomers. Compoundsdescribed herein may contain an asymmetric center and may thus exist asenantiomers. Where the compounds according to the invention possess twoor more asymmetric centers, they may additionally exist asdiastereomers. The present invention includes all such possiblestereoisomers as substantially pure resolved enantiomers, racemicmixtures thereof, as well as mixtures of diastereomers. The formulas areshown without a definitive stereochemistry at certain positions. Thepresent invention includes all stereoisomers of such formulas andpharmaceutically acceptable salts thereof. Diastereoisomeric pairs ofenantiomers may be separated by, for example, fractional crystallizationfrom a suitable solvent, and the pair of enantiomers thus obtained maybe separated into individual stereoisomers by conventional means, forexample by the use of an optically active acid or base as a resolvingagent or on a chiral HPLC column. Further, any enantiomer ordiastereomer of a compound of the general formula may be obtained bystereospecific synthesis using optically pure starting materials orreagents of known configuration.

As used herein, the term, “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event occurs and instances where it does not.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, rash, or gastric upset.In a preferred embodiment, the therapeutic composition is notimmunogenic when administered to a human patient for therapeuticpurposes.

“Providing” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted.

As used herein “subject” or “patient” refers to an animal or mammalincluding, but not limited to, human, dog, cat, horse, cow, pig, sheep,goat, chicken, monkey, rabbit, rat, mouse, etc.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. The methods herein for use contemplate prophylactic use aswell as curative use in therapy of an existing condition.

The terms “therapeutically effective” or “effective”, as used herein,may be used interchangeably and refer to an amount of a therapeuticcomposition embodiments of the present invention. For example, atherapeutically effective amount of a composition comprising an analogueof palmarumycin is a predetermined and an amount calculated to achievethe desired effect, i.e., to effectively inhibit Trx1/TrxR redox systemin an individual to whom the composition is administered.

The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,excipient, carrier, or vehicle.

Pentacyclic palmarumycins are structurally unique natural products withboth antifungal and antibacterial activities, but their anti-neoplasticeffects are not well established. Compounds such as auranofin,palmarumycin CP₁, 1,3-bis(2-chloroethyl)-1-nitrosourea, and AW464 havebeen shown to inhibit either thioredoxin, thioredoxin reductase, orboth. Embodiments of the invention relate to analogues of thepalmarumycin family. Further embodiments relate to the ability of thepalmarumycin analogues to inhibit the thioredoxin-thioredoxin reductase(Trx-1/TrxR redox) system. These analogues are tested for their abilityto inhibit tumor growth in vivo with and their use as a therapeuticagent. Additionally, embodiments provide use of palmarumycin analoguesthat inhibit Trx1/TrxR redox system to be used as lead compounds for thedevelopment of other therapeutic agents.

Methods for the inhibition of Trx1/TrxR redox system are alsocontemplated. Inhibition of Trx1/TrxR redox system may lead toinhibition of tumor growth, with the subsequent development oftherapeutic agents. Inhibition of Trx1/TrxR redox system may also leadto inhibition of cellular transcription factors, providing therapeuticcompounds, or lead compounds for the discovery of therapeutic compounds,for the treatment of medical conditions such as diabetic neuropathy,Sjogren's syndrome, HIV-AIDS, rheumatoid arthritis, reperfusion injuryor uncontrolled proliferation, as exemplified by cancer.

Since its discovery in the early 1960s, the thioredoxin—thioredoxinreductase system has been the subject of intense pharmacologicalstudies. The two redox active proteins have been isolated from manyspecies, and their medical interest is based in part on their value asindicators of widespread diseases such as rheumatoid arthritis, AIDS,and cancer. The cytosolic 12 kDa thioredoxin-1 (Trx-1) is the majorcellular protein disulfide reductase and its dithiol-disulfide activesite cysteine pair (CXXC) serves as an electron donor for enzymes suchas, but not limited to, ribonucleotide reductase, methionine sulfoxidereductase, and transcription factors including NF-κβ and theRef-1-dependent AP-1. Therefore, thioredoxin-1 is important for cellularredox regulation, signaling, and regulation of protein function, as wellas defense against oxidative stress and control of growth and apoptosis.

Thioredoxin-1 acts in concert with the glutathione—glutathione reductasesystem but with a rate of reaction orders of magnitude faster.Eukaryotic thioredoxin reductases (TrxR) are 112-130 kDa,selenium-dependent dimeric flavoproteins that also reduce substratessuch as hydroperoxides or vitamin C. These reductases containredox-active selenylsulfide-selenolthiol active sites and are inhibitedby aurothioglucose and auranofin. NADPH serves as reducing agent ofthioredoxin by thioredoxin reductase.

Pathophysiological effects of Trx-1/TrxR are indicated by Trx-1overexpression in human tumors such as, but not limited to, lung,colorectal and cervical cancers and leukemia. Secreted Trx-1 stimulatescancer cell growth and decreases sensitivity to induced apoptosis. TheTrx-1/TrxR system is therefore an important target for chemotherapeuticintervention. Although inhibitors of TrxR such as auranofin andnitrosoureas are quite effective, the search for new, more specific, andless toxic compounds is ongoing.

Embodiments of the invention provide new chemical compounds that inhibitthe activity of the Trx-1/TrxR redox system. Such compounds may beuseful as therapeutic agents, pharmacological probes, and/or leadcompounds for the development of therapeutic agents. These compounds mayinclude inhibitors of the thioredoxin—thioredoxin reductase system whichare less toxic than currently available Trx-1/TrxR redox inhibitingcompounds.

In one embodiments of the present invention, a compound, or saltthereof, having the general formula:

wherein R₁ may be H or OH;

R₂ may be OH, OCH₃, O(CH₂)nCH₃, OCH(CH₃)CH₂nCH₃, wherein n is 1-4,OCH₂CH₂-morpholino, OC(O)CH₂NH₂, OC(O)CH(CH₃) NH₂, OC(O)CH(CH(CH₃)₂)NH₂,OC(O)CH(CH₂Phenyl)NH₂, OC(O)CH(CH₂p-OHPhenyl)NH₂

OC(O)CH(CH₂OH)NH₂, OC(O)CH(CH₂SH)NH₂, OC(O)CH(CH₂COOH)NH₂,OC(O)CH(CH₂CH₂COOH)NH₂, OC(O)CH(CH₂CONH₂)NH₂, OC(O)CH(CH₂CH₂CONH₂)NH₂,OC(O)CH(CH(CH₃)CH₂CH₃)NH₂, OC(O)CH(CH2CH(CH₃)₂)NH₂, orOC(O)CH(CH(OH)CH₃)NH₂;

and

R₃ may be a hydrogen, NHNHC(CH₃)₂CONH₂ or a carbon-carbon bond, with theproviso that if R₁ is OH and R₃ is a carbon-carbon bond, R₂ is not OH;or salts thereof is provided. Exemplary salts include, but are notlimited to HCl, TFA, tosylate or any other pharmaceutically acceptablesalt. Such compounds may be useful as therapeutic agents orpharmaceutical compositions that optionally contain a pharmaceuticalexcipient or carrier.

In an alternative embodiment provided is a compound having the followingstructure:

In another alternative embodiment provided is a compound having thefollowing structure:

Preferred embodiments relate to a compound of the structure:

In a preferred embodiment, a salt of a palmarumycin compound has thestructure:

Other embodiments include compounds of the structures:

Preferred salts of the foregoing compounds include the following:

Each of the foregoing compounds may be useful as therapeutic agents,pharmaceutical compositions or diagnostic agents.

Another embodiment of the present invention provides a method ofinhibiting a thioredoxin/thioredoxin reductase system comprisingadministering an effective amount of a palmarumycin analog having thegeneral formula:

wherein R₁ may be H or OH;

R₂ may be OH, OCH₃, O(CH₂)nCH₃, OCH(CH₃)CH₂nCH₃, wherein n is 1-4,OCH₂CH₂-morpholino, OC(O)CH₂NH₂, OC(O)CH(CH₃) NH₂, OC(O)CH(CH(CH₃)₂)NH₂,OC(O)CH(CH₂Phenyl)NH₂, OC(O)CH(CH₂ p-OHPhenyl)NH₂

OC(O)CH(CH₂OH)NH₂, OC(O)CH(CH₂SH)NH₂, OC(O)CH(CH₂COOH)NH₂,OC(O)CH(CH₂CH₂COOH)NH₂, OC(O)CH(CH₂CONH₂)NH₂, OC(O)CH(CH₂CH₂CONH₂)NH₂,OC(O)CH(CH(CH₃)CH₂CH₃)NH₂, OC(O)CH(CH2CH(CH₃)₂)NH₂, orOC(O)CH(CH(OH)CH₃)NH₂;

and

R₃ may be a hydrogen, NHNHC(CH₃)₂CONH₂ or a carbon-carbon bond, with theproviso that if R₁ is OH and R₃ is a carbon-carbon bond, R₂ is not OH;or salts thereof. Exemplary salts include, but are not limited to HCl,TFA, tosylate or any other pharmaceutically acceptable salt.

In another embodiment, methods of inhibiting thioredoxin reductase-1 byadministering a palmarumycin analog described herein is provided. Inanother embodiment, such compounds may be useful in methods of treatingdiseases associated with the overexpression of thioredoxin-1, including,but not limited to, cancer, increased cell proliferation, and apoptosisby administering a therapeutically effective amount of a palmarumycinanalog as described herein.

The compounds may be administered in an effective amount to a subject inneed of such treatment. As such, the compounds described herein may beuseful for the treatment of cancer and other proliferative disorders.Administration of the compounds, in the form of a therapeutic agent, maybe carried out using oral, enteral, parenteral or topicaladministration, including, for example, intravenous, oral, transdermalor any other modes of administration optionally with a pharmaceuticalexcipient.

Pharmaceutical compositions can be used in the preparation of individualdosage forms. Consequently, pharmaceutical compositions and dosage formsof the invention may comprise the active ingredients disclosed herein(i.e., thioredoxin/thioredoxin reductase inhibitors, preferablypalmarumycin analogs, more preferably a O-glycyl naphthoquinonespiroketal (PX-916)). Further embodiments of the invention may compriseany therapeutic compound and/or therapeutic regiment which is to beassessed for its efficacy in inhibiting a tumor. Pharmaceuticalcompositions and dosage forms of the invention can further comprise oneor more excipients.

Single unit dosage forms of the invention are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), or transdermal administration to a patient. Examples ofdosage forms include, but are not limited to: tablets; caplets;capsules, such as soft elastic gelatin capsules; cachets; troches;lozenges; dispersions; suppositories; ointments; cataplasms (poultices);pastes; powders; dressings; creams; plasters; solutions; patches;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphoussolids), that can be reconstituted to provide liquid dosage formssuitable for parenteral administration to a patient.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage form usedin the acute treatment of a disease may contain larger amounts of one ormore of the active ingredients it comprises than a dosage form used inthe chronic treatment of the same disease. Similarly, a parenteraldosage form may contain smaller amounts of one or more of the activeingredients it comprises than an oral dosage form used to treat the samedisease. These and other ways in which specific dosage forms encompassedby this invention will vary from one another will be readily apparent tothose skilled in the art. See, e.g., Remington's PharmaceuticalSciences, 18th ed., Mack Publishing, Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms. The suitability of aparticular excipient may also depend on the specific active ingredientsin the dosage form. For example, the decomposition of some activeingredients may be accelerated by some excipients such as lactose, orwhen exposed to water. Active ingredients that comprise primary orsecondary amines are particularly susceptible to such accelerateddecomposition.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers,” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the invention comprise anamount preferably in a range from about 0.05 mg/kg/day to about 5,000mg/kg/day, more preferably in a range from about 0.5 mg/kg/day to about500 mg/kg/day, more preferably in a range of about 1 mg/kg/day to about50 mg/kg/day, and more preferably yet, the therapeutically effectiveamount is in a range from about 2 mg/kg/day to about 30 mg/kg/day.

The compounds of the invention are preferably administered in effectiveamounts. An effective amount is that amount of a preparation that alone,or together with further doses, produces the desired response. This mayinvolve only slowing the progression of the disease temporarily,although preferably, it involves halting the progression of the diseasepermanently or delaying the onset of or preventing the disease orcondition from occurring. This can be monitored by routine methods.Generally, doses of active compounds would be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses ranging from 20-500mg/kg will be suitable, preferably intravenously, intramuscularly, orintradermally, and in one or several administrations per day.

In general, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect for each therapeuticagent and each administrative protocol, and administration to specificpatients will be adjusted to within effective and safe ranges dependingon the patient condition and responsiveness to initial administrations.However, the ultimate administration protocol will be regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition and size of the patient, the compoundpotencies, the duration of the treatment and the severity of the diseasebeing treated. For example, a dosage regimen of the palmarumycinanalogs, preferably an O-glycyl naphthoquinone spiroketal (PX-916), canbe oral administration of from 1 mg/kg to 2000 mg/kg/day, preferably 1to 1000 mg/kg/day, more preferably 50 to 600 mg/kg/day, in two to four(preferably two) divided doses, to reduce tumor growth. Intermittenttherapy (e.g., one week out of three weeks or three out of four weeks)may also be used.

In the event that a response in a subject is insufficient at the initialdoses applied, higher doses (or effectively higher doses by a different,more localized delivery route) may be employed to the extent that thepatient tolerance permits. Multiple doses per day are contemplated toachieve appropriate systemic levels of compounds. Generally, a maximumdose is used, that is, the highest safe dose according to sound medicaljudgment. Those of ordinary skill in the art will understand, however,that a patient may insist upon a lower dose or tolerable dose formedical reasons, psychological reasons or for virtually any otherreason.

The development of therapeutics typically begins with the identificationof an active, or lead, compound that exhibits some of the propertiesrequired for safe and effective therapeutic intervention. Compounds withimproved properties are subsequently derived through iterative cycles ofanalog preparation and testing. Lead compounds are often identifiedusing high throughput screening (HTS), whereby large librariescontaining about tens to thousands or more compounds are tested usingrelatively simple assays to measure inhibition of processes critical tothe target indication, in this case inhibition of the Trx1/TrxR redoxsystem. Typically this means using biochemical assays to measure thefunction of one or more macromolecular targets.

All lead compounds share a common property, in the present invention toinhibit the Trx1/TrxR redox system. Screening by assaying for theinhibition of the Trx1/TrxR redox system identifies a small subset ofcompounds which can be further studied. All compounds from the originallibrary should be identified. Therefore, when multiple biochemicalactivities of the target (the Trx1/TrxR redox system) are known, all theactivities of each compound can be measured separately without theprohibitive effort that may be needed to screen the entire library usingmultiple functional assays.

HTS permit screening of large numbers (i.e., tens to thousands or more)of compounds in an efficient manner. Automated and miniaturized HTSassays are particularly preferred. HTS assays are designed to identify“hits” or “lead compounds” having the desired inhibitory property, fromwhich modifications can be designed to improve the desired property.Chemical modification of the “hit” or “lead compound” is often based onan identifiable structure/activity relationship between the “hit” andone or more of the palmarumycin analogues.

There are a number of different libraries used for the identification ofspecific small molecule inhibitors, including, (1) chemical libraries,(2) natural product libraries, and (3) combinatorial libraries comprisedof random peptides, oligonucleotides or organic molecules.

Chemical libraries consist of structural analogs of known compounds orcompounds that are identified as “hits” or “leads” via natural productscreening. Natural product libraries are derived from collections ofmicroorganisms, animals, plants, or marine organisms which are used tocreate mixtures for screening by: (1) fermentation and extraction ofbroths from soil, plant or marine microorganisms or (2) extraction ofplants or marine organisms. Natural product libraries may includemetabolites of a microorganism such as fungal metabolites, for example.Combinatorial libraries are composed of large numbers of peptides,oligonucleotides or organic compounds as a mixture. They are relativelyeasy to prepare by traditional automated synthesis methods.

Example 1

Various analogues of palmarumycin CP₁, were tested against two humanbreast cancer cell lines and several members displayed potent effects ininhibiting cell proliferation. A second generation series ofpalmarumycin CP₁ analogues showed increased in vitro activity, butfailed to reduce tumor growth in vivo. This lack of correlation betweenenzyme and animal assays may be attributed to the low water solubilityand limited bioavailability of the natural product lead structure(palmarumycin CP₁). Polar prodrug molecules with improved solubility andantitumor activity were then synthesized.

Typical solubilizing functions in prodrug derivatives include, but arenot limited to, phosphonate or phosphate esters, amino acid esters,phenolic acetates, and various other acyl groups. Starting with thenaphthoquinone spiroketal scaffold 5, (FIG. 1) the phenolic groups wereused to introduce a charged, hydrolytically cleavable function. Couplingof compounds 5 and 6 (herein referred to as PX-960) with variousBoc-protected amino acids proceeded in good yield and highregioselectivity (FIG. 1). Only the phenolic hydroxy group distal fromthe carbonyl functionality in 6 (PX-960) was acylated under the DCCmediated esterification conditions. This regioselectivity can beattributed to strong hydrogen bonding between the phenol and thecarbonyl oxygen as well as the inductive attenuation of thenucleophilicity at this site. Following esterification, the carbamatewas removed with 20% trifluoroacetate (TFA) in dichloromethane to afforda series of TFA salts which were tested for their ability to inhibitthioredoxin reductase along with general cytotoxicity.

In addition to amino esters, the introduction of a tertiary amine in theform of a morpholine heterocycle was also investigated (FIG. 2). Onceagain, selective monoetherification was observed at the hydroxyl groupdistal from the carbonyl group. Compounds 13 and 14 (FIG. 2) were alsosubjected to biological evaluation.

As expected, all naphthoquinone spiroketal prodrugs 10-14 (FIGS. 1-2)showed fundamentally equivalent low micromolar IC₅₀ values for MCF-7cell growth inhibition compared to the parent phenol 6 (PX-960) (Table1). In vitro inhibition of the thioredoxin-thioredoxin reductase systemwas more variable, and none of the prodrugs accomplished the nanomolarlevel of activity of PX-960, the parent compound, with glycyl prodrug 12(herein referred to as PX-916) being the notable exception. Differentamounts of the active drug may be released from the prodrug during theenzyme assays by spontaneous hydrolysis. However, prodrug PX-916 wasconsiderably more water soluble (0.7 mg/mL vs<0.1 mg/mL for PX-960) thanthe parent active compound, and furthermore use of 20% β-cyclodextrinincreased its solubility to 2 mg/mL. The half-life for the conversion ofPX-916 to PX-960 in ethanol at room temperature was t_(1/2)>5d. In waterat room temperature and pH4, PX-916 had a t_(1/2)=37 h, but it wasrapidly broken down at pH 7 and above (t_(1/2)<1 h). The relativelability of the prodrug at alkaline pH does not present a problem forformulation since pH 4 media can readily be administered to patients.

The chemical stability of PX-916 in mouse plasma at room temperature wast_(1/2)<2 minutes, and the prodrug was indeed converted to PX-960according to HPLC analysis. Upon release from its O-acyl protectivefunction, 6 (PX-960) had a t_(1/2)˜31 min in plasma. Accordingly,glycine-derivative PX-916 met all the requirements that were set forthfor further development as a lead compound in in vivo tumor xenograftmodels.

TABLE 1 IC₅₀ values [μM] for TrxR inhibition and human breast cancercell growth inhibition. TrxR inhibitory MCF-7 growth Entry Compoundactivity inhibition 1  6 (PX-960) 0.20 2.6 2 10 1.8 2.4 3 11 0.62 2.2 412 (PX-916) 0.28 3.1 5 13 1.6 1.2 6 14 4.2 2.6

Because of the promising biological profile of prodrug PX-916, theenantiomers of the spirocycle PX-960 were resolved to test if theyexhibited differential biological activities. The most direct approachwould be a separation via chiral HPLC. The low solubility that plaguedthe biological testing of PX-960 limited the efforts towards chiralseparation on a multi-milligram scale. However, small quantities (˜1 mg)of enantiomerically pure PX-960 could be obtained with a Chiralcel AD-Hcolumn, and both enantiomers demonstrated comparable in vitro activity.Since the difference in the absolute configuration of PX-960 relates tothe spatial orientation of the naphthaline ketal, the lack ofenantioselectivity in the biological assay supports the hypothesis thatthis group is not primarily involved in any activity-determininginteractions. Accordingly, the naphthaline ketal represents a preferredsite for chemical changes that target the optimization ofphysicochemical properties.

The water soluble, reversible prodrug derivatives of potent inhibitorsof the thioredoxin-thioredoxin reductase system were synthesized in aconvergent fashion. The O-glycyl naphthoquinone spiroketal PX-916demonstrated equivalent biological activity compared to the previouslead structure PX-960 in the in vivo MCF-7 tumor model as well as in thethioredoxin reductase inhibition assay. Moreover, PX-916 had 1-2 ordersof magnitude improved aqueous solubility and, while stable at pH 4,rapidly released the active compound under physiological conditions.

General Procedure for Coupling Reactions.

To a partial suspension of spirocycle 6 (PX-960, 92 mg, 0.28 mmol) inCH₂Cl₂ (6 mL) was added N-(tert-butoxycarbonyl)glycine (58 mg, 0.33mmol), DCC (74 mg, 0.36 mmol) and DMAP (7 mg, 0.06 mmol). The reactionmixture was stirred at room temperature for 1 h as the starting materialgradually dissolved and a white precipitate was formed. The cloudysolution was filtered, rinsed with CH₂Cl₂ and concentrated under reducedpressure. The residue was purified by chromatography on SiO₂(Hexanes/EtOAc, 7.3) to afford 112 mg (83%) of4′-(N-tert-butoxycarbonylamino)acetic acid palmaruymycin CP₁, ester 9 asa yellow solid: Mp 182-185 (dec., EtOAc/Hexanes); IR 3375, 2980, 1772,1662, 1609, 1506, 1419 cm⁻¹.

Spectral Data for 9.

¹H NMR (300 MHz, CHCl₃) δ 12.15 (s, 1H), 7.65 (t, 1H, J=8.0 Hz),7.58-7.48 (m, 2H), 7.44 (d, 1H, J=7.5 Hz), 7.25 (d, 1H, J=8.4 Hz), 7.14(d, 1H, 18.4 Hz), 7.03 (d, 1H, J=7.8 Hz), 7.01 (d, 1H, J=10.5 Hz), 6.95(d, 1H, J=8.2 Hz), 6.37 (d, 1H, J=10.5 Hz), 5.26 (brs, 1H), 4.34 (d, 2H,J 5.7 Hz), 1.50 (s, 9H): ¹³C NMR (75 MHz, CHCl₃) 5 188.8, 169.5, 162.1,156.1, 147.6, 145.4, 141.1, 139.5, 138.8, 136.8, 130.2, 128.7, 127.4,120.0, 119.9, 119.5, 115.8, 114.0, 113.7, 111.0, 109.6, 93.4, 80.6,42.9, 28.6; MS (EI) m/z: (rel intensity) 416 ([M-OtBu]⁺, 48), 389 (15),332 (100); HRMS (EI) calcd for C₂₃H₁₄NO₇, (M-Ot-Bu) 416.0770, found416.0776.

General Procedure for Deprotection.

To a solution of glycine ester 9 (95 mg, 0.19 mmol) in CH₂Cl₂ (4 mL) wasadded trifluoroacetic acid (1 mL). The reaction mixture was stirred atroom temperature for 30 min, and concentrated under reduced pressure toafford 98 mg (100%) of 12 as a yellow solid: IR 3200, 1772, 1665, 1610,1420, 1205 cm⁻¹.

Spectral Data for 12.

¹H NMR (300 MHz, CD₃CN) δ 12.09 (brs, 1H), 7.70 (dd, 1H, J=8.4, 7.7 Hz),7.64 (dd, 1H, J=8.6, 1.1 Hz), 7.58 (dd, 1H, J=8.5 Hz, 7.4 Hz), 7.45 (dd,1H, J=7.7, 1.0 Hz), 7.33 (d, 1H, J=8.3 Hz), 7.14 (dd, 1H, J=8.4, 1.0Hz), 7.07 (dd, 1H, J=7.3, 1.0 Hz), 7.06 (d, 1H, J=10.5 Hz), 7.00 (d, 1H,J 8.3 Hz), 6.35 (d, 1 H, J=10.5 Hz), 4.30 (s, 2H); ¹³C NMR (75 MHz,CD₃CN) δ 189.8, 167.4, 162.7, 160.8, (q, J=37.4 Hz), 148.5, 146.6,141.5, 140.6, 139.7, 137.8, 130.9, 129.9, 128.0, 121.0, 120.6, 120.4,117.1 (q, J=287.6 Hz), 116.4, 114.7, 114.4, 111.9, 110.4, 94.4, 41.8; MS(ESI) m/z (rel intensity) 390 ([M-OCOCF₃]⁺, 100) 359 (47); HRMS (ESI)calcd for C₂₂H₁₆NO₆ (M-OCOCF₃) 390.0978, found 390.0975.

General Procedure for Attachment of Morpholine Tether.

To a solution of spirocycle 6 (30 mg, 0.090 mmol) in THF (2 mL) wasadded N-(2-hydroxyethyl)morpholine (11 μL, 0.090 mmol), PPh₃, (26 mg,0.10 mmol) and DIAD (20 μL, 0.10 mmol). The reaction mixture was stirredat room temperature for 3 h then concentrated under reduced pressure.The residue was purified by chromatography on SiO₂(EtOAc/MeOH, 19:1) toafford 22 mg (55%) of 14 as a yellow solid film:

Spectral Data for 14.

¹H NMR (300 MHz, CHCl₃) δ 12.17 (s, 1H), 7.90 (d, 1H, J 8.5 Hz), 7.66(t, 1H, J 8.0 Hz), 7.50-7.45 (m, 2H), 7.14 (dd, 1H, J=8.4, 0.9 Hz), 7.04(d, 1H, J=7.3 Hz), 7.00 (d, 1H, J=10.5 Hz), 6.89 (d, 1H, J=8.3 Hz), 6.80(d, 1H, J=8.3 Hz), 6.35 (d, 1H, J 10.5 Hz), 4.30 (t, 2H, J=5.5 Hz),3.80-3.75 (m, 4H), 2.98 (t, 2H, J=5.5 Hz), 2.70-2.65 (m, 4H, J); MS(ESI), m/z (rel intensity) 446 ([M+1]⁺, 100), 359 (65), 331 (20), 272(12); HRMS (ESI) calcd for C₂₆H₂₄NO₆ (M+H) 446.1604, found 446.1581.

Spectral Data for 10.

¹H NMR (300 MHz, CD₃CN) δ 8.11 (dd, 1H, J=7.8, 1.1 Hz), 8.00 (dd, 1H,J=7.8, 0.9 Hz), 7.84 (td, 1H, J=7.6, 1.4 Hz), 7.71 (td, 1H, J=7.6, 1.2Hz), 7.68-7.57 (m, 2H), 7.36 (d, 1H, J=8.3 Hz), 7.09 (dd, 1H, J=7.3, 1.0Hz), 7.08 (d, 1.H, J=10.6 Hz), 7.02 (d, 1H, J=8.3 Hz), 6.38 (d, 1H,J10.6 Hz), 4.28 (s, 2H); MS (ESI) m/z (rel intensity) 374 ([M-OCOCF₃]⁺,37), 317 (100), 299 (30); HRMS (ESI) calcd for C₂₂H₁₆NO₅ (M-OCOCF₃)374.1028, found 374.1034.

Spectral Data for 11.

¹H NMR (300 MHz, CD₃CN) δ 8.09 (dd, 1H, J=7.8, 1.3 Hz), 7.96 (d, 0.5H,J=7.8 Hz), 7.94 (d, 0.5H, J=7.8 Hz), 7.79 (t, 1H, J=7.6 Hz), 7.69 (d,1H, J=7.7 Hz), 7.64 (d, 1H, J 8.5 Hz), 7.55 (t, 1H, J=8.1 Hz), 7.33 (d,1H, J=8.2 Hz), 7.06-7.00 (m, 2H), 6.97 (dd, 1H, J=8.2, 1.7 Hz), 6.33 (d,0.5H, J=10.6 Hz), 6.31 (d, 0.5H, J=10.6 Hz), 4.44 (d, 1H, J=4.2 Hz),2.65-2.59 (m, 1H), 1.22 (d, 6H, J=6.9 Hz); MS (ESI) m/z (rel intensity)416 ([M-OCOCF₃]⁺, 100), 307 (12), 225 (18), 199 (18); HRMS (ESI) calcdfor C₂₅H₂₂NO₅ (M-OCOCF₃) 416.1498, found 416.1485.

Spectral Data for 13

¹H NMR (300 MHz, CHCl₃)δ8.18 Hz (dd, 1H, J=7.8, 1.2 Hz), 7.98 (d, 1H,J=7.8 Hz), 7.90 (d, 1H, J=8.3 Hz), 7.77 (td, 1H, J=7.5, 1.3 Hz), 7.67(td, 1H, J7.5, 1.1 Hz), 7.48 (t, 1H, J=8.1 Hz), 7.03 (d, 1H, J=7.0 Hz),7.01 (d, 1H, J=10.5 Hz), 6.89 (d, 1H, J=8.3 Hz), 6.80 (d, 1H, J=8.3 Hz),6.39 (d, 1H, J=10.5 Hz), 4.30 (t, 2H, J=5.6 Hz), 3.80-3.75 (m, 4H), 2.97(t, 2H, J=5.6 Hz), 2.70-2.65 (m, 4H); MS (ESI) m/z (rel intensity) 430([M+1]⁺, 100), 343 (12), 279 (8); HRMS (ESI) calcd for C₂₆H₂₄NO₅ (M+H)430.1654, found 430.1568.

Retention times for the two 6 (PX-960) enantiomers on an AD-H column in14% i-PrOH/Hexanes were 8.27 min and 10.07 min, respectively.

Stability.

PX-916 was stable as a stock solution in ethanol at room temperaturewith a half life of >5 days. However, in 0.1M sodium phosphate bufferPX-916 showed pH dependent degradation with a half life at pH 4.0 of 37hr, at pH 7.0 a half life of 1 hr and at pH 10.0 a half life of <1 hr.Therefore, for in vitro studies PX-916 was used as a stock solution inethanol, and for in vivo studies made fresh in pH 4.0 buffer vehicle.

Inhibition of TR.

PX-916 was a potent inhibitor of purified human TR with an IC₅₀ of 0.28μm which is similar to that of palmarumycin (Table 2). However, unlikepalmarumycin which is almost insoluble in aqueous media, PX-916 issoluble with an apparent maximum solubility in water of around 10 mg.Based upon the observation that PX-916 was rapidly converted to PX-960in aqueous solution, the ability of PX-960 to inhibit purified human TRwas measured and found to be similar to that of PX-916 (Table 2). PX-916was a selective inhibitor of human TR compared to two other NADPHdependent reductases with a selectivity of at least 200 for humanglutathione reductase and human cytochrome P-450 reductase (Table 3).

TABLE 2 Inhibition of thioredoxin reductase and cell growth bypalmuramaycin analogs Inhibition of human Inhibition of MCF-7 breastthioredoxin reductase cancer cell growth Compound IC₅₀ (μM) IC₅₀ (μM)palmarumycin C1 0.35 1.0 PX-911 3.2 9.2 PX-916 0.28 3.1 PX-960 0.27 4.1

TABLE 3 Selectivity of PX-916 for TR compared to other human reductaseshuman reductase Inhibition (source) IC₅₀ (μM) Selectivity thioredoxinreductase 0.28 (placenta) glutathione reductase >50 >200 (red bloodcell) cytochrome P-450 reductase 29.2 104 (recombinant)

In Vitro Activity.

Cell growth inhibition of MCF-7 human breast cancer cells bypalmarumycin, PX-916 and PX-960 occurred at similar concentration of 1to 3 μM (Table 2). MCF-7 human breast cancer cells were grown in mediumcontaining 1 μM selenium (Se) for 7 days which increased cellularthioredoxin reductase activity by about 5 fold as previously reported.When the cells were exposed to 1 μM PX-916 there was a time dependentinhibition of cellular TR that was maximum at 24 hr (FIG. 3A). The IC₅₀for inhibition of cellular TR by PX-916 was 0.25 μM and maximuminhibition occurred at 0.5 μM (FIG. 3B). Thus, inhibition of purifiedhuman TR, MCF-7 cellular TR and cell growth inhibition of MCF-7 cells byPX-916 occurred at about the same concentrations.

In Vivo Inhibition of Tumor TR and Antitumor Activity.

A single intravenous (i.v.) dose of PX-916 of 25 mg/kg inhibited MCF-7human tumor xenograft TR up to about 60% at 24 hr and the inhibition wasmaintained for at least 48 hr (FIG. 3C). The growth of A-673 humanrhabdomyosarcoma xenografts (±SE, n=6 mice) was decreased from 153±35mm³/d in the vehicle controlto 5±3 mm³/d for 5 days after dosing withPX-916 at 30 mg/kg/d ip for five doses (97% inhibition; P>0.01) (FIG.4A). PX-916 administered i.v. showed good antitumor activity against theSHP-77 small cell lung cancer with a decrease in tumor growth rate 5days after the end of dosing (±SE, n=8 mice) from about 150±48 mm³/dayin vehicle control to 27 mm³/day when administered at 25 mg/kg i.v.daily for 5 doses (82% inhibition; P<0.05) (FIG. 4B). In this study 3 ofeight nice had no histologically detectable tumor when the study wasterminated on day 42. Tumor growth was decreased to about 91±24 mm³/day(39% inhibition, P<0.05) by PX-916 administered i.v. at 10 mg/kg i.v.daily for 8 doses. The growth of MCF-7 human breast cancer xenograftswas decreased 5 days after the end of dosing from 47+10 mm³/day in thevehicle control to 22±4 mm³/day by PX-916 at 27.5 mg/kg i.v. daily for 5doses (52% inhibition, P<0.05), 22±8 mm³/day by PX-916 at 27.5 mg/kgi.v. every other day for 5 doses (52% inhibition, P>0.05) and to 18.5±8mm³/day by PX-916 at 27.5 mg/kg orally for 5 doses (62% inhibition,P<0.05) (FIG. 4C).

Tumor HIF-1α and VEGF.

Levels of the HIF-1α transcription factor and its downstream target VEGFare increased by thioredoxin-1 expression. We examined the effect ofPX-916 administration on tumor HIF-1α and VEGF levels (FIG. 5A).Twenty-four hours after the last dose of five daily doses of PX-916 of25 mg/kg, there was a decrease in MCF-7 xenograft staining for bothHIF-1α and VEGF. At the same time, levels of tumor thioredoxin reductaseactivity were decreased by 75% (FIG. 5B).

Toxicity.

A single i.v. dose of PX-916 of 50 mg/kg to female scid mice was lethal.However, female scid mice tolerated 5 daily doses of PX-916 of 25 mg/kgi.v. The major toxicities observed 24 hr after the last dose wasneutropenia and thrombocytopenia, with no observable increase in plasmaliver enzymes and no significant weight loss (Table 4).

TABLE 4 Toxicity of PX-916 in scid mice. PX-916 was administered tofemale scid mice at 25 mg/kg iv daily for 5 days and micewere killed 24hr after the last dose. There were 4 mice per group and values are themean ± SE. ALT AST WB NE LY MO RBC Hb PLT Schedule Dose Δbodywtg U/I U/IK/μl K/μl K/μl K/μl M/μl g/dl K/μl Control −1.2 ± 1.5 30.3 ± 10   154.1± 62.4 3.0 ± 0.6  2.5 ± 0.5  0.4 ± 0.1 0.11 ± 0.05 9.4 ± 0.5 15.5 ± 0.7773 ± 67  QD × 5 iv 25 −0.6 ± 0.3 39.8 ± 12.8 166.9 ± 29.8 1.3 ± 0.6*0.9 ± 0.3* 0.3 ± 0.1  005 ± 0.01 8.9 ± 0.4 14.9 ± 0.4 436 ± 142* *= p <0.05

Pharmacokinetics.

When incubated with fresh mouse plasma at room temperature, PX-960 had ahalf life of about 31 minutes at room temperature while PX-916 rapidlydisappeared and was converted to PX-960 with a half life of less than2.0 minutes. When PX-916 was administered to mice at 25 mg/g i.v., itcould not be detected in plasma 5 minutes after administration and onlya very small peak of the parent compound PX-960 could be detected at 5minutes. No PX-960 was detected at 30 minutes. Two metabolite peakscould be seen at 5 minutes, but were undetectable by 30 minutes.

PX-916 was synthesized as a water soluble prodrug of the palmarumycinanalog PX-960 and was found to retain the ability to inhibit purifiedTrxR with an IC₅₀ of 0.28 μM. PX-916 also inhibited TrxR activity inMCF-7 human breast cancer cells with an IC₅₀ of 0.25 μM and was aninhibitor of MCF-7 cell growth with an IC₅₀ of 3.1 μM. PX-916 was anNADPH and time dependent, apparently, irreversible inhibitor ofthioredoxin reductase-1, most likely reacting with theselenocysteine-containing catalytic site. Two other NADPH dependentreductases, human glutathione reductase and cytochrome P-450 reductase,were not inhibited by PX-916 until concentrations were increased togreater than 100 fold higher.

Stability studies showed that at physiological pH PX-916 was stable forabout 1 hr and slowly converted to its parent PX-960 with a half life of1 hr. It was much more stable at pH 4.0 and was formulated at this pHfor i.v. administration. In mouse plasma the breakdown of PX-916 toPX-960 was rapid with a half life less than 2 minutes. Thus, theinhibition of TrxR in tumor xenografts and the antitumor activity islikely to be almost completely due to the parent PX-960. PX-960 could bedetected only transiently in mouse plasma after administration of PX-916due to rapid metabolism or likely rapid distribution of the verylipophilic PX-960. Thus, PX-916 provides a novel soluble and stableprodrug for the administration of PX-960.

When a single dose of 25 mg/kg was administered to mice with MCF-7breast cancer xenografts the tumor TrxR activity was inhibited by up to60% and remained inhibited for 48 hr. Repeated administration of PX-916for five (5) days gave 75% inhibition of tumor thioredoxin reductase 24hrs after the last dose.

PX-916 given i.p. or i.v. showed antitumor activity against A673rhabdomyosarcoma, SHP-77 small cell lung cancer, and MCF-7 breastcancer. In SHP-77, complete tumor regressions were seen in some mice.The most active schedule was every other day administration, and tumorgrowth was inhibited as long as the drug was given. Significantantitumor activity was not seen following oral administration at dosesthat gave i.v. antitumor activity. Thioredoxin-1 acts by a redoxmechanism to increase HIF-1 a levels and VEGF formation, associated withan increase in tumor angiogenesis, and this effect was reversed by athioredoxin-1 inhibitor. MCF-7 tumor xenografts in mice treated withPX-916 showed a decrease in tumor HIF-1 a and VEGF presumably due to theinhibition of thioredoxin-1 redox signaling. Inhibition of thioredoxinreductase-1 might affect other pathways in the cell. A recent studyusing small interfering RNA to knockdown thioredoxin reductase-1expression and microarray analysis showed changes in leukotriene B412-hydroxydehydrogenase, ubiquitin D, differentiation enhancing factor,fibronectin 1, apolipoprotein 3, prosaposin, choline/ethanolaminephosphotransferase, and IFN-a-inducible protein genes.

PX-916, a water-soluble prodrug of a palmarumycin CP1 analogue, rapidlyreleases the parent compound at physiologic pH and in plasma, but isstable at acid pH, allowing its i.v. administration. PX-916 is aninhibitor of purified human thioredoxin reductase-1 and of thioredoxinreductase activity in cells and tumor xenografts when given to mice.PX-916 exhibited antitumor activity against several animal tumor models,with some cures, and blocked the expression of the downstream targets ofthioredoxin-1 signaling, HIF-1 a and VEGF, in the tumors.

Example 2

Further palmarumycin analogs were synthesized and tested. The followinganalogs were synthesized: eee269-II, eee86-III, eee263-II and eee273-II.The stability of the analogs was measured in various solutions. Thestability of 100 g/ml of compound in a 0.1M sodium phosphate buffer ispresented in Table 5 below.

TABLE 5 Compound pH T_(1/2) eee269-11 7.0 12 min 4.0 >100 hr eee86-1117.0 >100 hr 4.0 >100 hr eee262-11 7.0 >100 hr 4.0 >100 hr eee273-117.0 >100 hr 4.0 >100 hr

The stability of the analogs in ethanol is presented in FIG. 6. Thestability of the analogs and formation of the parent compound in plasmawas measured and is presented in FIG. 7, FIG. 8 and Table 6 (measured bythe formation of parent compound over the first 20 min of incubation at1 mg/ml and at 33° C.), below. As shown below, eee273-II appears veryinsoluble at 1 mg/ml, but not studied in mouse plasma and eee269-II wasnot studied in plasma because it is unstable in aqueous solutions.

TABLE 6 Stability of palmarumycin analog prodrugs in plasma. Mouse ES1emouse Human Plasma plasma plasma area/20 min area/20 min area/20 minSML-216 350 210 135 eee86-III 20 17 70 eee263-II 42 82 81 eee273-II 19

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontained within this specification.

1-15. (canceled)
 16. A compound, or salt thereof, having the generalformula:

wherein R₁ is H or OH; R₂ is selected from the group consisting ofO(CH₂)nCH₃, OCH(CH₃)CHnCH₃, OCH₂CH₂-morpholino, OC(O)CH₂NH₂, andOC(O)CH(CH(CH₃)₂)NH₂, OC(O)CH(CH₃)NH₂, OC(O)CH(CH₂Phenyl)NH₂,OC(O)CH(CH₂ p-OHPhenyl)NH₂

OC(O)CH(CH₂OH)NH₂, OC(O)CH(CH₂SH)NH₂, OC(O)CH(CH₂COOH)NH₂,OC(O)CH(CH₂CH₂COOH)NH₂, OC(O)CH(CH₂CONH₂)NH₂, OC(O)CH(CH₂CH₂CONH₂)NH₂,OC(O)CH(CH(CH₃)CH₂CH₃)NH₂, OC(O)CH(CH₂CH(CH₃)₂)NH₂,OC(O)CH(CH(OH)CH₃)NH₂; and

n is 1-4, and R₃ is hydrogen, or NHNHC(CH₃)₂CONH₂,

represents the option of having a double bond.